Capillary electrophoresis system

ABSTRACT

A capillary electrophoresis system includes a capillary reservoir. The capillary reservoir includes a capillary tip flow chamber configured to receive respective capillary tips and to conduct fluid past the capillary tips, and an electrode flow chamber in which an electrode is disposed and configured to conduct fluid past the electrode, the electrode flow chamber being separate from and in fluid communication with the capillary tip flow chamber. An ultraviolet (UV) light absorbance-based multiplexed capillary electrophoresis system includes a first enclosure and a second enclosure. The first enclosure covers a UV light source, and includes a slit. The second enclosure covers the first enclosure, a collimating lens, and a capillary window.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/677,535, filed Aug.15, 2017; which is a continuation-in-part of U.S. Ser. No. 14/822,956filed Aug. 11, 2015, now abandoned; which is a continuation of U.S. Ser.No. 13/470,870, filed May 14, 2012, now U.S. Pat. No. 9,140,666, issuedSep. 22, 2015; which claims priority to U.S. Provisional application61/643,411, filed May 7, 2012; and U.S. Ser. No. 13/470,870 is also acontinuation-in-part of U.S. Ser. No. 29/421,549, filed Mar. 15, 2012,now U.S. Pat. No. D689,621, issued Sep. 10, 2013; all of which areherein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to a system for multi-channel capillaryelectrophoresis.

Description of Related Art

The current next-generation sequencing (NGS) platforms use a variety oftechnologies for sequencing, including pyrosequencing, ion-sequencing,sequencing by synthesis, or sequencing by ligation. Although thesetechnologies have some minor variations, they all have a generallycommon DNA library preparation procedure, which includes genomic DNAquality & quality assessment, DNA fragmentation and sizing (involvingmechanical shearing, sonication, nebulization, or enzyme digestion), DNArepair and end polishing, and a last step of platform-specific adaptorligation. With a rapidly growing demand for DNA sequence information,there is a critical need to reduce the time required for the preparationof DNA libraries.

A labor-intensive step in DNA library preparation is the qualification(size determination) and quantification of both un-sheared genomic DNAand downstream fragmented DNA. Existing methods for DNA fragmentanalysis include agarose gel electrophoresis, capillary electrophoresis,and chip-based electrophoresis. Agarose gel electrophoresis is laborintensive, requiring gel preparation, sample transfer via pipetting, andimage analysis. The images obtained by agarose electrophoresis are oftendistorted, resulting in questionable or unreliable data. It isimpossible to use agarose gel electrophoresis for accuratequantification of DNA, which means that a separate, second method (UV orfluorescence spectroscopy) is required for quantification. Finally,agarose gel electrophoresis is difficult to automate. Chip or micro-chipbased electrophoresis provides an improvement in data quality overagarose gel electrophoresis but is still labor intensive. For example,chip-based methods require manual steps to load gel, markers andsamples. Even though these microchip or chip based electrophoresis unitscan run a single sample in seconds or minutes, the sample and gelloading are barriers to ease-of-use, especially when running hundreds orthousands of samples. Also, existing chip-based systems are unable toquantify genomic DNA. Capillary electrophoresis (CE) offers advantagesover both agarose electrophoresis and microchip electrophoresis in thatgel-fill and sample loading is automated.

Multiplex capillary electrophoresis is known. For example, Kennedy andKurt in U.S. Pat. No. 6,833,062 describe a multiplex absorbance basedcapillary electrophoresis system and method. Yeung et al. in U.S. Pat.No. 5,324,401 describe a multiplex fluorescent based capillaryelectrophoresis system. Although these systems offer the advantage ofanalyzing multiple samples simultaneously, and can run several platessequentially, they lack the ability to load or change multiple sampleplates while the system is running, and they also lack a simple workflowfor efficient sample analysis.

While existing commercial CE systems can be automated with a roboticsystem, stand-alone systems are not fully automated or lack thesensitivity and data quality required for adequate DNA library analysis.An example of a CE instrument with a robot-capable interface is given byKurt et al. in U.S. Pat. No. 7,118,659. For the construction of DNAlibraries, as well as other applications such as mutation detection, itis often necessary to run thousands of samples per day, but theimplementation of a robotic system for sample handling is prohibitivelyexpensive, and many labs lack the expertise necessary for themaintenance and operation of sophisticated robotic systems. Automatedforms of micro-slab-gel electrophoresis have been developed, such asthose described in United States Patent Application number 20100126857.These allow for automatic analysis of multiple samples, but thetechniques either still require significant human intervention, or theydo not have the throughput required for high-volume applications.Amirkhanian et al. in U.S. Pat. No. 6,828,567 describe a 12-channelmultiplex capillary electrophoresis system capable of measuring up 12samples at a time using multiplex capillary electrophoresis. However,this system is not capable of measuring multiple 96-well plates, anddoes not have the workflow that allows the analysis of thousands ofsamples per day.

As can be seen, there a need for an automated capillary electrophoresissystem that a) eliminates the complexity, cost, and required expertiseof a robotic system b) enables users to run from one to several thousandsamples per day and c) allows users to conveniently load several platesor samples onto a capillary electrophoresis system while the system isrunning other samples and d) has the small size and footprint of astand-alone capillary electrophoresis unit.

This invention has as a primary objective the fulfillment of the abovedescribed needs.

BRIEF SUMMARY OF THE INVENTION

The present invention is an ultraviolet light absorbance-based multiplexcapillary electrophoresis system and console with an improved samplehandling and control method for the analysis of samples.

One embodiment of the invention is a console with a series of at leastfour and preferably at least six vertically stacked user-accessibledrawers that can each hold a plate containing from 1 to 384 samplewells. Preferably, each user accessible drawer holds a sample platecontaining 96 sample wells. The system is configured so that sampleplates can be loaded onto the system at any time, including during theelectrophoresis or analysis of samples. User “A” can walk up to themachine, load a row of 12 samples, enter loading and analysisinstructions onto the computer and walk away. While user “A” samples arerunning, user “B” can walk up to the machine, load a tray of 96 samples,enter loading and analysis instructions and walk away. User “C” can walkup to the machine, load 12 samples, while either user “A” or user “B”samples are running, enter loading and analysis instructions, and walkaway. Two of the preferred six user-accessible drawers are used to holdan electrophoresis run buffer and a waste tray.

Another embodiment of the invention is a mechanical stage thattransports sample trays and/or buffer or waste trays from any one of thevertically stacked user-accessible drawers to the injection electrodesand capillary tips of the multiplex capillary array of the capillaryelectrophoresis subsystem.

Another embodiment of the invention uses a computer program that enablesa user to create a queue of jobs, with each job representing an analysisof a new set of samples. This computer system enables users to enter jobdata even when the system is running samples. For example, user “A”loads “sample plate 1” into the system into Drawer 3 and uses a computerprogram to add a job to a queue, the job representing the injection andcapillary electrophoresis of samples in “sample plate 1” in Drawer 3.While the system is running user A's samples, user B loads plate 2 intoDrawer 4 and uses the same computer program to add a job to a queue, thejob representing the injection and capillary electrophoresis of samplesin “sample plate 2” in Drawer 4. User C loads “sample plate 3” intoDrawer 5 and uses the same computer program to add a job to the queue,the job representing the injection and capillary electrophoresis ofsamples in “sample plate 3” in Drawer 5.

Another embodiment of the invention is an ultraviolet absorbance basedcapillary electrophoresis system comprising a console housing anoperable multiplexed capillary electrophoresis system; a capillary arraycontaining at least 12 capillaries; a UV light source; a first enclosurecovering said UV light source, wherein said first enclosure contains aslit; and a second enclosure covering said first enclosure, acollimating lens, and capillary window of said capillary array.

Yet another embodiment of the invention is a capillary electrophoresissystem comprising a console housing an operable multiplexed capillaryelectrophoresis system and a reservoir containing a flow channel forpassing a conductive fluid past the capillary tips of a capillary arrayand a second, separate channel for passing the same conductive fluidpast an electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a left-front-view of the instrument, with 6 drawers forholding sample and buffer plates.

FIG. 2 shows a right-front view of the instrument with one drawer pulledout for placement of a buffer plate and the top and side doorcompartments open.

FIG. 3 shows the x-z stage assembly.

FIG. 4 shows a drawer, stage assembly, tray holder, and sample plate.

FIG. 5 shows the bottom of a tray holder.

FIG. 6 shows a right-side view of the instrument without the cover.

FIG. 7 shows the left-side view of the instrument without the cover.

FIG. 8 shows a capillary array cartridge.

FIG. 9 shows the flow-chart for the software control program forcreating a queue of jobs.

FIG. 10 shows a computer screen image of the computer software.

FIG. 11 shows the positioning of a sample plate under the array by thestage.

FIG. 12A shows a view of the capillary electrophoresis reservoir system.

FIG. 12B shows a view of the capillary electrophoresis reservoir system.

FIG. 13A shows a view of the x-z stage relative to the drawers.

FIG. 13B shows a view of the x-z stage with a sample tray lifted.

FIG. 14A shows an alternate electrophoresis system with an optics pathcover.

FIG. 14B shows an alternative electrophoresis system with the opticspath cover removed.

FIG. 14C shows an alternative electrophoresis system with a cut-out viewof the optics path cover.

FIG. 15A shows an alternate electrophoresis reservoir system.

FIG. 15B shows a side view of an alternate electrophoresis reservoirsystem.

FIG. 16A shows a light-source box with a slit cover.

FIG. 16B shows a light-source box with the slit removed.

FIG. 17 shows a sliding-interlocking capillary array window mountingmechanism.

FIG. 18A show an alternate capillary array cartridge design.

FIG. 18B shows an adaptor for mounting a capillary array window to thearray base plate.

FIG. 19A shows an electropherogram run obtained with the electrophoresissystem of the invention.

FIG. 19B shows another electropherogram run with use of an enclosure ofFIG. 14A.

FIG. 19C shows an electropherogram run with a slit and enclosure of FIG.14A removed.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a multiplexed capillary electrophoresis system withenhanced workflow. The capillary electrophoresis system and apparatus ofthe present invention includes an absorbance or fluorescence-basedcapillary electrophoresis sub-system with a light source, a method forcarrying light from the light source to the sample windows of amultiplex capillary array containing at least 12 capillaries (preferably96 capillaries), and a method for detecting light emitted (fluorescence)or absorbed (absorbance) from the sample windows of a multiplex array.The sub-system also includes a method for pumping buffers and gelsthrough the capillaries, as well as a method for application of anelectric field for electrophoretic separation. The optics of thefluorescent-based sub system of the present invention are described byPang in United States Patent Applications 20070131870 and 20100140505,herein incorporated by reference in their entirety. The optics of anapplicable absorbance-based system, as well as the fluid handling,reservoir venting, application of electric field, and selection offluids via a syringe pump and a 6-way distribution valve are discussedby Kennedy et al. in U.S. Pat. Nos. 7,534,335 and 6,833,062, hereinincorporated by reference their entirety.

Referring to FIG. 1 the multiplex capillary system (or unit orinstrument) and/or console 16, with enhanced workflow has a door 10 foreasy access to the loading of gels, two drawers 11 for the easy loadingof a buffer tray and a waste tray. Drawers 12 can be opened for easyloading of 96-well PCR plates, tube strips, vials, or other samplecontainers. A top door 13 can be opened to access a replaceablecapillary array, array window, and capillary reservoir. An indicatorlight 14 is used to for notifying users of the active application of ahigh-voltage for electrophoresis. A removable back-panel 15 allowsaccess to electronics such as a high-voltage power supply, electricalcommunication panels, a pump board, pressure transducer board, and stagedriver electronics. The back panel 15 also allows maintenance access tothe x-z stage, which is used to move sample trays from the drawers 11and 12 to a capillary array.

FIG. 2 shows the multiplex capillary system used with the enhancedworkflow console 16 of FIG. 1 with the top and side doors open. Areplaceable capillary array (cartridge) 17 holds either 12 or 96capillaries for multiplex capillary electrophoresis. An LED light guide67 guides light from a LED engine located in the back compartment to thearray window block 22 which is inserted between the array window holder19 and LED light guide and window holder 18. In this view, array windowblock 22 is attached to the capillary array 17 for display. When thecapillary array 17 is removed from the system 16, the array window block22 can be attached to the capillary array 17 (as shown). When thecapillary array 17 is fully installed, the array window block 22 is notvisible because it is sandwiched between the array window holder 19 andLED light guide and window holder 18. A vent valve 21 is connected tothe top of a capillary reservoir 20. A syringe pump 23 coupled with a6-way distribution valve 29 delivers fluids and electrophoresis gelsfrom fluid containers 24 and 25 into the capillary reservoir 20, wastecontainer 26, or capillaries in the capillary array 17. A fan 27 is usedfor forcing cool air from the back compartment through the capillaryarray 17, past the outside of the capillary reservoir 20, down past thefluid containers 24, 25 and finally out the bottom of the instrument.LED indicator lights 120 are used to indicate the presence or absence oftrays in the drawers. A buffer tray 28 is shown in a drawer (11, FIG.1). The capillary array reservoir tip 91 is shown inserted into thecapillary reservoir 20.

The concepts and practical implementation of motion control systems areknown. For example, Sabonovic and Ohnishi; “Motion Control” John Wileyand Sons, 2011, herein incorporated by reference in its entirety,discusses practical methods for the design and implementation of motioncontrol. It does not, however, show an enhanced CE workflow console 16as depicted here.

FIG. 3 shows the x-z stage assembly 48, which is used to transportsample plates or trays (50, FIG. 4) and associated tray holders (51,FIG. 4) from the drawers (12 FIG. 1) to the injection capillaries (72,FIG. 8) and injection electrodes (71, FIG. 8) of the capillary array(17, FIG. 8). The x-z stage assembly 48 is also used to position abuffer tray or waste tray (28, FIG. 2) from the drawers (11, FIG. 1) tothe injection capillaries 72 and injection electrodes 71 of thecapillary array 17 (FIG. 8). The x-z stage assembly 48 has a traycarrier 31 with alignment pins 32, which align with holes (57, FIG. 5)on the bottom of the tray holder (51, FIG. 4) to prevent subsequentsliding or movement of the tray holders 51 during transport. Aprotective cover 34, made of metal or plastic, is used to prevent gelsor other liquids from spilling onto the x-direction guide rails 38 andx-direction drive belt 37 of the x-z stage assembly 48. An x-drivestepper motor 35 is used as the electro-mechanical driver for motion inthe x-direction. A drive pulley 36 is attached to the stepper motor 35and x-direction drive belt 37 which drives the stage carrier 39 back-andforth along the guide-bars 38. A second drive pulley (not shown) is usedon belt 37 towards the back-end of the stage, which allows the belt 37to make a full loop when affixed to stage carrier 39. Any motor-inducedmovement of the belt 37 induces an x-direction movement of the stagecarrier 39 on the guide rails 38. A stepper-motor for the z-position islocated at 41, which is attached to a drive pulley/belt configurationsimilar to that shown in the x-direction. The z-direction drive belt isshown as 43. The z-position motor/pulley/belt is used to move the traycarrier 31 up and down the guide bars 40. Top plate 33 serves as astructural support for the guide bars 40. An electrical communicationstrip 44 is used to communicate between an electrical motor controlboard 46 and the stepper motors 41 and 35. An x-direction membranepotentiometer strip 49, along with appropriate control electronics, isused to determine and control the absolute position of the stage carrier39 in the x-direction. A z-direction membrane potentiometer strip 42,along with appropriate control electronics, is used to determine theabsolute position of the tray carrier 31 in the z-direction. Linearencoders or rotational encoders (on the stepper motor 35, 41) arealternative forms of positional measurement and control. Bearings 45 arelocated on each guide bar 40 and guide rail 38 to enable friction-freemovement of both the tray carrier 31 and the stage carrier 39. Note thatthere are two guide bars 40 or guide rails 38 per axis. Electrical cordguide straps 47 are attached to a back support, which also holds theelectrical control board 46 for the x-z stage assembly 48.

FIG. 4 shows a drawer 12, superimposed on an image of the x-z stageassembly 48, tray holder 51, and 96-well sample tray 50. The tray holder51 is molded to specifically hold a 96-well plate, shown here as 50.Alternative moldings of the tray holder 51 allow for different sampleplates. Holes (57, FIG. 5) on the bottom of the tray holder 51 alignwith the alignment pins 32 of the tray carrier (31 FIG. 4). Notches 53in the tray holder 51 align with alignment pins 52 on the drawer 12 toenable the tray holder 51 to fit in a tight, reproducible way within thesample drawer 12.

FIG. 6 Shows a right-side view of the electrophoresis system, with achassis 66, pump motor and control system 61, pump control board 62, LEDlight engine 69, LED light line 67, high voltage power supply board 65,capable of applying 0.0 kV to 15 kV across the electrodes of thecapillary array 17, a CCD camera 64, capillary array cartridge 17, arraywindow holder 19, capillary reservoir 20, drawers 11, drawers 12, fluidlines 68, waste container 26, gel containers 25 and syringe 23. A USBelectronic distribution board is shown as 63.

FIG. 7 shows a left side-view of the electrophoresis unit 16 showing thex-z stage assembly 48, which moves tray holders 51 and sample trays orplates 50 from a drawer 12 or 11 to the bottom of the capillary array17. The stage unit 48 can move the sample tray holder 51 and sample tray50 up in the z-direction to lift the tray holder/sample tray off of thedrawer 12 (or 11), move back in the x-direction away from the sampledrawers 12, and then move the sample plate 50 up in the z-direction tothe bottom of the capillary array 17. After electrokinetic orhydrodynamic injection, the stage unit 48 can move the sample trayholder/sample tray back down to the target drawer position (down in thez-direction), move forward in the x-direction just above the sampleplate 50, and then drop down in the z-direction to set the sample trayholder/sample tray onto the drawer 12. When the sample tray holder 51 isresting in a drawer 12, the back edge of the sample tray holder 51 andsample tray 50 are aligned so that they do not lie directly underneaththe capillary array 17. This allows the sample stage tray carrier (31,FIG. 3) to move up and down along the entire z-axis with a trayholder/sample tray without colliding into other tray holders/sampletrays in the drawers 12. The alignment pins (70, FIG. 8) on the bottomof the capillary array 17 are used to align the tray holder 51 with asample tray 50 so that the capillary and electrode tips (the tips of theinjection capillaries 72 and the injection electrodes 71) dip into eachsample well of the sample plate 50 and do not collide with other areasof the sample plate 50. This is shown in more detail in FIG. 11, whichshows a sample tray holder 51 with a sample tray 50 aligned underneath acapillary array 17. Alignment holes 56 on the tray holder 51 force thealignment of the tray holder 51 with the capillary array alignment pins70.

FIG. 7 also shows high voltage power supply board 65 and high voltagepower supply cable 75 (to the capillary array 17).

FIG. 8 shows a capillary array cartridge 17, with rigid plastic supportstructure 77, window storage and transport screw 80, capillary supportcards 76, high voltage power supply cable 75, and insulating supportstructure or load header 73 onto which the electric circuit board 74 isplaced.

Electrodes 71 protrude through the electric circuit board 74, throughthe insulating support structure or load header 73, and protrude throughthe bottom of the capillary array 17. The electrode material isstainless steel or tungsten. The electrode dimension, which is not acritical aspect of the invention, is 50 mm length times 0.29 mmdiameter. The protrusion from the bottom of the cartridge base is 20.0mm. The electrodes 71 are soldered onto the circuit board 74. The highvoltage power supply cable 75 is also soldered to the same circuit ofthe electrical circuit board 74, which enables contact of the electrodes71 with the high voltage power supply (65, FIG. 6). Capillary tips (thetips of the injection capillaries 72) are threaded through the electriccircuit board 74 and insulated support structure or load header 73 andare aligned immediately adjacent and parallel to the electrode tips (thetips of the injection electrodes 71). The distance between the capillarytips and electrode tips are from 0.1 mm to 4 mm. The ends of thecapillary tips and the ends of the electrode tips lie in a single plane(i.e. the capillary tips and electrode tips are the substantially thesame length, with length variation of no more than about +/−1 mm).Preferably, the length variation of capillary tips and electrode tips isless than 0.5 mm. The capillaries 72 thread through the bottom of thecapillary array 17, through the insulating support structure or loadheader 73, through the electric circuit board 74, through the capillarysupport cards 76 (which are supported by the rigid plastic supportstructure 77) through the capillary window holder 78 with capillarywindows 79 centered in the opening of the window holder 78, and thenfinally through the capillary reservoir tip 91, in which all capillaries72 (in this case 12) are threaded through a single hole. For 96capillary arrays 17, capillaries 72 are threaded in groups of 12, 8, 4,or 2, (preferably 4) in the capillary reservoir tip 91. The capillaries72 are held in place in the capillary reservoir tip 91 with an adhesive,such as a thermally or UV-curable epoxy. The tips of the capillaries 72located at the capillary reservoir tip 91 may be referred to as firstcapillary tips, and the tips of the capillaries 72 located at theelectrodes 71 (at the ends of the capillaries 72 opposite to the firstcapillary tips and capillary reservoir tip 91) may be referred to assecond capillary tips.

FIG. 12A shows the capillary reservoir 20, with a reservoir body(indicated by the arrow of 20), capillary reservoir tip 91, slider bar130 (for locking capillary reservoir tip 91 into the capillary reservoir20, through alignment of a notch on the capillary reservoir tip 91 andthe slider bar 130), vent block valve 21, waste tube out 138, wasteblock valve 132, and pressure transducer cavity 133.

FIG. 12B shows an alternate cut-out view of the capillary reservoir 20,with the reservoir body, capillary reservoir tip 91, slider bar 130,vent block valve 21, waste tube out 138, waste block valve 132,electrode for attachment to ground (or ground electrode) 135, pressuretransducer cavity 133, pressure transducer 136, pressure transducercable for attachment to analog/digital board 137, and fluid tube input134 (from syringe pump 23, FIG. 2).

The reservoir body of the capillary reservoir 20 can be made of anysolid material such as acrylic, Teflon, PETE, aluminum, polyethylene,ABS, or other common metals or plastics. The key criterion is that thematerial is durable and chemically resistant to the materials used. Apreferred material is acrylic or Teflon.

FIG. 13A shows the x-z stage unit 48 in relation to the drawers 11 and12. The x-z stage is located directly behind the drawers 11 and 12, andcan move the stage carrier (39, FIG. 13B) back-and forth in thex-direction using the stepper-motor for the x-position (35, FIG. 3). Asample tray 50 is removed from a drawer 12 (or 11) by first moving thestage forward, towards the drawers 11 and 12, in the x-direction. Thetray carrier (31, FIG. 3) lifts a tray holder up 51 and off a drawer 12in the z-direction using the z-direction stepper motor (41, FIG. 3). Thestage carrier 39 is then moved back in the x-direction, away from thedrawers 11 and 12, as shown in FIG. 13B. The stage carrier 39 is thenmoved up in the z-direction to move the tray holder 51 and sample tray50 to the injection position of the capillary array 17 (FIG. 11).

A typical strategy for pumping fluids for capillary electrophoresis isas follows. Consider the following 6 positions of the six-waydistribution valve (29, FIG. 2) on the syringe pump 23. Position 1 isconnected to the bottom of the capillary reservoir 20 (fluid tube input134, FIG. 12B); position 2 is connected through a tube to a bottle ofconditioning fluid (a fluid for conditioning the walls of thecapillaries 72); position 3 is connected to a “Gel 1” which is used forthe analysis of genomic DNA, position 4 is connected to a “Gel 2” whichis used for the analysis of fragmented DNA, position 5 is unused, andposition 6 is connected to the waste bottle 26.

Step A: The capillary reservoir 20 is first emptied by opening position1 (reservoir), filling the syringe 23 with fluid that is in thecapillary reservoir 20, closing position 1, opening position 6, andemptying fluid to the waste (waste container 26). This is repeated untilthe capillary reservoir 20 is empty. Block valves 21 and 132 are keptopen during this process to enable efficient draining of the capillaryreservoir 20.

Step B: The capillary reservoir 20 is then filled with conditioningsolution by opening position 2, filling the syringe 23 with conditioningsolution, closing position 2, opening position 1, and filling thecapillary reservoir 20 with conditioning solution. Block valve 21 isclosed, but block valve 132 to waste (waste container 26) is open,enabling the over-filling of the capillary reservoir 20 withconditioning solution.

Step C: The capillaries 72 are filled by closing both vent block valve21 and waste vent valve 132. The syringe 23 is filled with capillaryconditioning solution. Position 1 is opened, and fluid is pressurefilled through the capillaries 72 at a minimum of 100 psi for apre-determined time, which may range from 1 minute to 20 minutes.

Step D: The capillary reservoir 20 is emptied by step A, and thenre-filled with gel using the same process as in Step B, except thatposition 3 for the gel is used on the 6-way distribution valve 29.

Step E: The capillaries 72 are filled with gel using a process analogousto Step C.

After steps A-E, the capillaries 72 are ready for electrophoresis.

A general strategy and process for analyzing samples usingelectrophoresis is as follows.

Samples are placed into a 96-well plate (sample tray or plate 50) foranalysis. The user places the sample plate 50 into a sample drawer (12,FIG. 1), and then adds jobs to a computer-based queue, corresponding tothe analysis of a specific row or the entire sample plate 50 in thedrawer 12.

The computer, which is the control system of the instrument, executesthe analysis of the row or entire sample tray 50 of interest.

A key embodiment of the invention is the workflow of the capillaryelectrophoresis system 16. Drawers (11, FIG. 1) allow easy placement ofbuffer and waste trays 28 into the system 16. Drawers (12, FIG. 1) alloweasy placement of sample trays 50 into the system 16. Of particularimportance is the ability to place or remove sample trays 50 fromdrawers (12, FIG. 1) while the system 16 is performing capillaryelectrophoresis. Indicator lights (120, FIG. 1) show if a tray 28, 50 ispresent or absent in a drawer 11, 12, which let users know if a drawer11, 12 is in place. A typical workflow for a 12-capillary multiplexsystem is as follows: User A walks up to the machine with sample tray 1,and places it into the third drawer from the top (one of drawers 12,FIG. 1). User “A” then fills a queue with three jobs, which correspondto performing capillary electrophoresis on the three rows of samples:sample tray 1 row A, sample tray 1 row B, and sample tray 1 row C. User“A” then instructs the computer to execute the queue, and as a result,the system begins capillary electrophoresis of sample tray 1, row A, andwill continue executing jobs in the queue until there are no more jobs.User “B” then comes up and places sample tray 2 into the fourth drawerfrom the top (one of drawers 12, FIG. 1). User “B” then adds 8 jobs tothe queue corresponding the performing of capillary electrophoresis on 8rows of samples: sample tray 2, rows A-H. The computer will continueanalyzing user “A” samples until they are finished, and then continue onwith the analysis of user “B” samples. In the meantime, user “C” walksup and loads sample tray 3 into the fifth drawer from the top (one ofdrawers 12, FIG. 1). User “C” then adds 1 job to the queue correspondingto the analysis of 1 row of samples: sample tray 3, row A. This processcan continue indefinitely, as long as there is sufficient gel in gelcontainers (25 in FIG. 2), or if there is sufficient run buffer in thebuffer tray (28, FIG. 2) located in top drawer 11, FIG. 1. It is, amongother things, the enabling of this workflow, via the drawers 11 and 12,sample stage (x-z stage assembly 48), and computer program with a queuefor loading jobs that differentiates the present invention from theprior art systems for CE workflow.

A computer program enables users to load a sample plate 50 into thedesired vertical drawer (12, FIG. 1), and instruct the system 16 to runthe desired rows or entire sample plate 50, while the system 16 isrunning other samples. This allows multiple users to load samples and/orsample plates 50, or a single user to load multiple samples and/orsample plates 50 without first having to wait for the electrophoresis ofother samples to be complete.

FIG. 9 shows the general flow diagram of the work process and computerprogram. A user loads a sample tray 50 into a drawer (12, FIG. 1) of thesystem 16. On the computer, user then selects the sample tray 50, editssample names and/or tray name. User further selects or defines a method(time of separation, electric field used for separation, gel selection,etc.). This selected sample tray 50, along with an associated method isdefined as a “job”, which is then placed into a queue. The computer asan instrument control device, fetches jobs from the queue, and controlsthe instrument system 16) for every task, including operation of thesyringe pump 23, operation of the high voltage power supply 65, and themotion control stage (48, FIG. 3). For each run (or job), there may be avariety of tasks, with each task requiring direct command and control ofsubunits of the system 16. Tasks associated with control of the syringepump 23 include emptying/filling the capillary reservoir 20 withconditioning fluid, forcing conditioning fluid through the capillaries72, emptying/filling the capillary reservoir 20 with gel, and forcinggel through the capillaries 72. Tasks associated with control of the x-zstage assembly 48 may include moving or removing a waste tray to/fromthe inlet side of the capillaries 72 and electrodes 71 of the capillaryarray 17, moving or removing a buffer tray 28 to/from the side of thecapillaries 72 and electrodes 71 of the capillary array 17, ormoving/removing a sample tray 50 to/from the side of the capillaries 72and electrodes 71 of the capillary array 17. Tasks associated withcontrol of the high voltage power supply 65 include turning off/on ahigh voltage for capillary electrophoresis separation. Other tasks areassociated with the camera 64 (acquisition of data), and block valves 21and 132. For each set of samples, the program will complete all tasksrequired to obtain a set of electropherograms. Once these tasks arecomplete, the program fetches another job from the queue. If the queueis empty, all sample runs are complete (until the user initiates anotherqueue).

The graphical result of this computer program is shown in FIG. 10, whichshows a list of samples to be analyzed in queue 101, an option to addrows or sample trays 50 to the queue 102, and an option to select thetray number for analysis 103. It is these three aspects that arecritical to software portion of the invention: a) Selection of tray 103(corresponding to a drawer 12 FIG. 1) b) Adding the sample set to aqueue (102, FIG. 10) and c) A queue of active samples for analysis (101,FIG. 10), which are executed in sequence until all jobs are complete.Another critical aspect is the ability to add samples to instrumentdrawers (11, 12, FIG. 1) and queue (101, FIG. 10) while the instrument(system 16) is running other samples.

An example of an electrophoresis system modified to operate withabsorbance-based detection is shown in FIGS. 14A-14C, 16A and 16B, whichin particular may be an ultraviolet (UV) light absorbance-basedmultiplexed capillary electrophoresis system. An absorbance-based systemincludes an ultraviolet (UV) or visible light source such as (but notlimited to) a mercury lamp, a zinc lamp, a Light-Emitting Diode,deuterium lamp, or tungsten lamp. The absorbance-based system includes afirst enclosure (or lamp housing) 1404 and a second enclosure 1401. Thesecond enclosure (1401 FIG. 14A) encloses and seals the light within thefirst enclosure (lamp housing) (1404 FIG. 14B) as well as a collimatinglens (1403 FIG. 14B) up to the capillary window 79, such that thecollimating lens 1403 is disposed between a UV light source 1603 in thefirst enclosure (lamp housing) 1404 and the capillary window 79. Thesecond enclosure 1401 (FIG. 14A) substantially blocks access of anyairflow to the optics train. An optional additional fan port with a fan(1402 FIG. 14A) is used to circulate air for cooling the capillaries(capillary bundle 1708, FIG. 17) close to the capillary reservoir (thecapillary reservoir 20 shown in FIGS. 2, 6, 12A and 12B, or thecapillary reservoir shown in FIGS. 15A and 15B). The UV light source1603 within the first enclosure (lamp housing) (1404 FIG. 14B) alongwith the collimating lens (1403 FIG. 14B) is used to direct and focusthe light onto the capillary array window 79. FIG. 14C shows a cutoutview of the second enclosure 1401, showing the first enclosure (lamphousing) 1404, collimating lens 1403, and capillary bundle 1708.

An alternate reservoir design is shown in FIG. 15A, with fluid ports1501, 1502, and 1503. Ports 1501 and 1502 may be used to pump fluidsinto and out of the capillary reservoir. Fluid may pass in a continuouspath from input port 1501, past (or through) tube 1507, to capillary tipflow chamber 1509, to electrode flow chamber 1504, and finally out thewaste port 1503 through waste valve 132. An alternate flow path is frominput port 1502, to electrode flow chamber 1504, and finally out thewaste port 1503 through waste valve 132. Note that the waste valve 132in this figure is incorporated into the reservoir block. Port 1503 isonly used as a waste port, and thus fluid always flows out of thecapillary reservoir through the waste valve 132. Also shown are apressure transducer 136, and an electrode cord for attachment to ground1508. The fluid path that runs past the (second) capillary tips 1511 isshown as a tube 1507 which widens into a capillary tip flow chamber1509. A second relatively large electrode flow chamber 1504 in fluidcommunication with and part of fluid path 1507 and capillary tip flowchamber 1509 is used to house and contain the (ground) electrode 1505(which is attached to electrode cord 1508). Any bubbles generated by theelectrode 1505 during electrophoresis are contained to the electrodeflow chamber 1504, and cannot easily reach the capillary tips 1511 orcapillary flow chamber 1509. The electrode flow chamber 1504 ispositioned such that the highest point, corresponding to the location ofthe pressure transducer 136 is lower than the capillaries (in particularthe capillary tips 1511) or lower than the lowest point in the capillaryflow chamber 1509. The electrode flow chamber 1504 has a diameter thatis at least 2× that of the general fluid flow path 1507. Prior toinitially performing a series of electrophoresis runs, a preferable stepis to pump gel from port 1501 past capillary tips 1511 through capillaryflow chamber 1509 through the waste valve 132 while port 1502 isblocked. For an intermediate purge of gel between runs, a preferableflow path is from port 1502 through port 1503 via waste valve 132 withport 1501 blocked. When ports 1502 and 1503 are blocked, gel or otherfluids are pressurized through capillary tips 1511 by applying fluidflow through port 1501 while monitoring pressure through pressuretransducer 136. The wide flow path of the electrode flow chamber 1504provides a relatively large gel volume in which the electrode 1505 forattachment to ground 1508 is affixed.

FIG. 15B shows a side-view of the capillary reservoir emphasizing therelative orientation of pressure transducer 136, pressure transducercable for attachment to analog/digital board 137 electrode chamber 1504,and electrode 1505.

FIG. 16A shows a close-up of the first enclosure (lamp housing) 1404,where a light source or lamp (1603, FIG. 16B) is fixed within a hollowrectangular box (designated by the lead line for 1404) covered with aplate or slit cover (1602) containing a slit (1604) allowing emission ofthe light to the capillary windows (79, FIG. 17) through the collimatinglens 1403. FIG. 16B shows lamp housing 1404 with plate 1602 removed,revealing lamp or light source 1603. A power cable for the lamp 1603extends from the bottom of lamp housing 1404. The width of the slit 1604within the plate (1604) may vary from 50 micrometers up to 4000micrometers, with a preferable slit width range of 500 to 1500micrometers.

FIG. 17 shows an alternate capillary array window block 1701, which ismounted into the electrophoresis system 16 by sliding the window holderslots 1702 over two guide posts 1703 onto instrument mount 1705. Thecapillary array window block 1701 snaps into place with aspring-pressure ball snap connector 1704. External pressure mount 1707affixes capillary bundle 1708 in the capillary array window block 1701and is held in place by adhesive, screws, or any other attachment means.A slit or mask 1706 which is described in U.S. Pat. No. 5,900,934(Gilby) is optionally placed in front of the capillary array or bundle1708 to limit or direct light from the light source to individualcapillaries.

FIG. 18A shows an alternate capillary array, with capillary array windowblock 1701 mounted for storage and transport to the insulating supportstructure or load header 73 with a window storage and transport screw(80, FIG. 8 and FIG. 18A) attached to single-piece bridging orconnecting piece 1801. The single bridging or connecting piece 1801 isnot attached to rigid frame 77 (FIG. 8 and FIG. 18A) but instead isattached to the insulating support structure or load header 73 by anadhesive, screw, or any other common method of attachment.

FIG. 18B shows single-piece bridging or connecting piece 1801, whichallows the capillary array window block 1701 to be mounted to theinsulating support structure or load header 73.

The following example is offered to illustrate certain aspects of theinvention without in any way limiting it.

EXAMPLE 1

A denaturing single-stranded oligomer gel or sieving matrix “DN-415”(available from Advanced Analytical Technologies, Inc.) was used forthis example. The “DN-415” sieving matrix was pumped into a plurality ofninety-six capillaries with an effective length of 55 centimeter (cm)and a total length of 80 cm (75 micron I.D.) using the capillaryelectrophoresis system described in this specification. An oligomer mixstandard, consisting of 10 micromolar each of poly-T oligomers 19-mer,20-mer, 39-mer, 40-mer, 59-mer, and 60-mer was used to evaluateseparation efficiency. The conditions used for obtaining theelectropherograms in this example are: 1) the gel-filled capillarieswere treated with an electrophoresis pre-run by applying 12 kV for 20minutes prior to injection of sample; 2) the oligomer mix standard wasinjected onto the capillary electrophoresis system (present invention)using an electrokinetic injection of 3 kV for 7 sec., and 3) this wasimmediately followed by an electrophoresis run using a constant appliedvoltage of 12 kV for 70 minutes. FIG. 19A shows an electropherogram runobtained with the electrophoresis system of the present invention withthe second enclosure 1401 (FIG. 14A) and lamp housing slit cover 1602(FIG. 16). Region 1901 of the electropherogram shows a low level ofbaseline noise. FIG. 19B shows an electropherogram run obtained with theelectrophoresis system of the present invention with second enclosure1401 (FIG. 14A) removed from the system and lamp housing slit cover 1602(FIG. 16) remaining on the system. Region 1902 in FIG. 19B of theelectropherogram shows a higher baseline noise and drift relative toregion 1901 in FIG. 19A. FIG. 19C shows an electropherogram run obtainedwith the electrophoresis system of the present invention with secondenclosure 1401 (FIG. 14A) removed from the system and lamp housing slitcover 1602 (FIG. 16) also removed from the system. Region 1903 in FIG.19C of the electropherogram shows a significantly higher baseline driftand noise relative to region 1901 in FIG. 19A. This example shows thatthe lamp housing slit cover 1602 and second enclosure 1401 are importantin obtaining electropherograms with low baseline noise and drift.

As can be seen from the above description, the system eliminates theneed for expensive robots, enables the user to run many samples per day,allows loading of new samples while running others, and yet has a smallsize footprint. Furthermore, the present invention enables the analysisof samples with high quality signal to noise, and a low level ofbaseline drift. It therefore fulfills the need described.

1. A capillary electrophoresis system, comprising: a console configuredto house a replaceable capillary array comprising a plurality ofcapillaries; a power supply disposed in the console and configured toapply a voltage across each of the capillaries effective for performingcapillary electrophoresis on a sample in one or more of the capillaries;and a capillary reservoir, comprising: a fluid input port; a capillarytip flow chamber configured to receive respective capillary tips of thecapillaries and to conduct fluid past the capillary tips; an electrode;an electrode flow chamber in which the electrode is disposed andconfigured to conduct fluid past the electrode, the electrode flowchamber separate from and in fluid communication with the capillary tipflow chamber; a waste port; and a tube defining a fluid flow path fromthe fluid input port to the capillary tip flow chamber, to the electrodeflow chamber, and to the waste port, wherein a highest point of theelectrode flow chamber is lower than a lowest point of the capillary tipflow chamber.
 2. The system of claim 1, comprising a detector disposedin the console and configured to detect light transmitted from thecapillaries.
 3. The system of claim 1, comprising a plurality of drawersdisposed in the console and configured to hold a plurality of multi-wellplates, each drawer movable between a closed position inside the consoleand an open position outside the console at which the drawer isexternally accessible.
 4. The system of claim 3, comprising a motioncontrol system disposed in the console and configured to move one ormore of the multi-well plates to and/or from one or more of therespective drawers.
 5. The system of claim 4, wherein the consolecomprises an injection position at which the replaceable capillary arrayis received, and the motion control system is configured to move the oneor more multi-well plates from the one or more respective drawers to theinjection position.
 6. The system of claim 5, comprising a controldevice programmed to control an operation of the multiplex capillaryelectrophoresis system, the operation comprising sequentially performingat least a first job and a second job according to a job queue inputtedby one or more users into the control device, wherein: the first jobcomprises operating the motion control system to move a first multi-wellplate loaded in the first drawer to the injection position, injecting afirst sample from the first multi-well plate into at least some of thecapillaries, and operating the power supply to apply the voltage toperform a first electrophoresis run on the first sample; the second jobcomprises operating the motion control system to move a secondmulti-well plate loaded in the drawer to the injection position, injecta second sample from the second multi-well plate into at least some ofthe capillaries, and operating the power supply to apply the voltage toperform a second electrophoresis run on the second sample; and thecontrol device is configured to control performing the firstelectrophoresis run while the second multi-well plate is being loadedinto the second drawer while the second drawer is in the open position.7. The system of claim 6, wherein the control device is configured tocontrol performing the first electrophoresis run while a user of the oneor more users is inputting the second job into the job queue.
 8. Thesystem of claim 4, wherein the motion control system comprises a traycarrier configured to support a selected multi-well plate of theplurality of multi-well plates.
 9. The system of claim 8, wherein themotion control system comprises an x-drive motor configured to drivemotion of the tray carrier back and forth relative to the plurality ofdrawers, and a z-drive motor configured to drive motion of the traycarrier up and down relative to the plurality of drawers.
 10. The systemof claim 8, wherein the tray carrier comprises one or more alignmentpins configured to engage a tray holder of the selected multi-wellplate.
 11. The system of claim 3, wherein each drawer comprises one ormore alignment pins configured to engage a tray holder of a selectedmulti-well plate of the plurality of multi-well plates.
 12. The systemof claim 1, wherein the capillary tips received in the capillary tipflow chamber are first capillary tips, the capillaries compriserespective second capillary tips opposite to the corresponding firstcapillary tips, and the system further comprises a plurality ofinjection electrodes communicating with the power supply and disposedproximate to the second capillary tips.
 13. The system of claim 1,comprising a control device programmed to control an operation of thesystem, the operation comprising: controlling the system to inject thesample into the replaceable capillary array; and controlling the powersupply to, after injection of the sample, apply the voltage.
 14. Thesystem of claim 13, comprising a motion control system disposed in theconsole and configured to move one or more of the multi-well plates toand/or from an injection position of the console, wherein the operationcontrolled by the control device further comprises: controlling themotion control system to, before the injecting, move a selected one ofthe multi-well plates to the injection position.
 15. The system of claim1, comprising: a UV light source; a first enclosure covering the UVlight source, wherein the first enclosure comprises a slit; acollimating lens configured to be disposed between the UV light sourceand a capillary window of the replaceable capillary array; and a secondenclosure configured to cover the first enclosure, the collimating lens,and the capillary window.
 16. An ultraviolet (UV) light absorbance-basedmultiplexed capillary electrophoresis system, comprising: a consoleconfigured to house a replaceable capillary array comprising a pluralityof capillaries; a power supply disposed in the console and configured toapply a voltage across each of the capillaries effective for performingcapillary electrophoresis on a sample in one or more of the capillaries;a UV light source; a first enclosure covering the UV light source,wherein the first enclosure comprises a slit; a collimating lensdisposed between the UV light source and the capillary window; and asecond enclosure disposed in the console and covering the firstenclosure, the collimating lens, and the capillary window.
 17. Thesystem of claim 16, wherein the slit is less than 1500 microns in width.18. The system of claim 16, comprising a detector disposed in theconsole and configured to detect light transmitted from the capillariesfor acquisition of absorbance data.